Insecticides of Plant Origin - ACS Publications - American Chemical

Chapter 3

Search for New Pesticides from Higher Plants 1,4

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A. Alkofahi , J . K. Rupprecht , J . E . Anderson , J . L . McLaughlin , K. L. Mikolajczak , and Bernard A. Scott 2

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Downloaded by CORNELL UNIV on May 11, 2012 | http://pubs.acs.org Publication Date: February 23, 1989 | doi: 10.1021/bk-1989-0387.ch003

Department of Medicinal Chemistry and Pharmacognosy, School of Pharmacy and Pharmacal Sciences, Purdue University, West Lafayette, IN 47907 Northern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, Peoria, IL 61604 Lilly Research Laboratories, Greenfield Laboratories, P.O. Box 708, Greenfield, IN 46140 2

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Pest control methods of the near future w i l l include potent, more selective, and bio-degradable pesticides discovered as the natural protectants of higher plants. A number of botanical pesticides (pyrethrins, rotenoids, nicotine, the natural isobutylamides, quassia, sabadilla, hellebore, and ryania) are enjoying expanding commercial uses. Some chemical companies are developing additional plant extracts from the Meliaceae and Rutaceae containing potent limonoids such as azadirachtin. Our laboratories have been involved i n p e s t i c i d a l screening programs of higher plant extracts and have already screened several hundred plant species against a battery of indicator pests. L i s t s of promising leads have been tabulated, and b i o a c t i v i t y - d i r e c t e d fractionations, using brine shrimp l e t h a l i t y as a simple in-house bioassay, are i n progress. Our first product i s asimicin, a novel acetogenin (polyketide) from the bark of the paw paw tree, Asimina triloba (Annonaceae); this group of very potent compounds now offers a host of chemical and b i o l o g i c a l challenges f o r further research and commercial exploitation. The demand for p e s t i c i d a l and herbicidal compounds to control animal and plant pests and weeds has created an agro-chemical business i n the U.S. which totals over $15 b i l l i o n annually. These synthetic wonders have facilitated astonishing gains i n agricultural production, but these same compounds, i n some cases, 4

Current address: Faculty of Pharmacy, Jordan University of Science and Technology, Irbid, Jordan

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0097-6156/89/0387-0025$06.00/0 1989 American Chemical Society

In Insecticides of Plant Origin; Arnason, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

INSECTICIDES OF PLANT ORIGIN


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have posed serious problems to health and environmental safety. Newer, more selective, and biodegradable compounds must replace these generally toxic and persistent chemicals of the present and the immediate past. Never pest control methods w i l l include: a) use of natural predators, parasites, and pathogens; b) breeding resistant varieties of crop species; c) pest sterilization techniques; d) use of mating and feeding attractants i n combination with traps; e) development of i n t e r f e r i n g hormones; f) improved methods for grain storage; and g) discovery and development of better, more s p e c i f i c , and biodegradable, pesticides (1,2). The expanding use of synthetic pyrethroids, which are based on natural prototypes from chrysanthemum flowers, i s a good example of the desired approach to better pesticides Q ) ; these compounds are more s p e c i f i c for k i l l i n g insects and exhibit few negative effects on plants, livestock, or humans; i n addition, there i s often less resistance developed to these compounds i n the target insects. A number of additional botanical insecticides (rotenoids, nicotine, the natural isobutylamides, quassia, sabadilla, hellebore, and ryania) are enjoying expanding commercial uses (4), and a number of chemical companies are evaluating (and i n some cases developing) extracts from plants of the Meliaceae and Rutaceae containing i n s e c t i c i d a l limonoids such as azadirachtin (J>,£) · To protect themselves from being eaten, plants have, i n e f f e c t , been waging biochemical warfare for thousands of years against insects and herbivores Q . â ) . Perhaps many plant species which f a i l e d to develop such protective "secondary metabolites" were consumed and made extinct; thus, the potential for finding pesticides i n higher plants should be high. The job of the phytochemist i s to detect, isolate, and i d e n t i f y these compounds. Martin Jacobson has spent much of h i s research career encouraging an expansion of research i n this area (1,4,7,8). The prospect of discovery and exploitation of these novel higher plant metabolites, useful as pesticides or pesticide prototypes, seems excellent and i s especially timely. For the past several years, our laboratories have been involved i n dedicated programs of bioactivity-directed screening and fractionation of higher plant extracts to y i e l d such agents. SCREENING HIGHER PLANTS FOR PESTICIDES The single, most important, factor i n the search for new bioactive substances i s the convenience and r e l i a b i l i t y of the bioassay systems. Screening bioassays must be inexpensive and rapid, have broad application to numerous target organisms, be reproducible and s t a t i s t i c a l l y v a l i d , and require very l i t t l e of the test substance. No single pest model meets a l l of these requirements, and a battery of indicator organisms from various taxa i s , undoubtedly, the best approach to screening. However, f i n a n c i a l constraints usually force the choice of one or only a few. EUROPEAN CORN BORER BIOASSAY AND THEVETIA THEVETIOIDES About ten years ago at the Peoria screening program for pesticides was

USDA laboratory, a plant I n i t i a t e d using 7-day-old



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ALKOFAHI

ET A L .

PesticidesfromHigher Plants

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larvae of the European c o m borer, Ostrinia nub Hal is (£) . This insect was chosen because of i t s economic importance to agriculture i n the midwest. Seeds of several dozen plant species, from the extensive USDA seed c o l l e c t i o n i n Peoria, were extracted i n a Soxhlet sequentially with hexane and ethanol, and the extract residues were incorporated into the corn borer diet. Mortalities were determined, generally after nine days. This screen revealed several species which seemed worthy of fractionation ( 1 0 ) . One of the most promising leads was the ethanol extract of the defatted seeds of Thevetia Chevetioides (HBK) K. Schum. (Apocynaceae). Large-scale extraction and fractionation of seeds from Mexico was laboriously monitored with the corn borer bioassay. Percent mortalities were determined for partitioned fractions and for pools of similar combined fractions from large chromatography columns. The active pools were rechromatographed and tested with the corn borer bioassay at every step, u n t i l two i n s e c t i c i d a l components were c r y s t a l l i z e d . Using spectral ( H nmr, C nmr, i r ) and physical (mp, t i c ) methods, the two compounds were i d e n t i f i e d as n e r i i f o l i n and 2 * - a c e t y l n e r i i f o l i n , cardioactive glycosides, previously known i n other species of the Apocynaceae ( H ) .

Ο

Ο* N e r i i f o l i n (corn borer L D 5 0 3 0 ppm i n the diet) was ca. s i x times as active as 2 ' - a c e t y l n e r i i f o l i n ( L D ^ Q 1 9 2 ppm) and showed excellent dose response curves with l a r v a l mortality. Under such test conditions, carbofuran, a popular commercial insecticide f o r corn borers, has an L D 5 0 of 1 - 2 ppm. Further testing of n e r i i f o l i n showed potent i n s e c t i c i d a l a c t i v i t i e s against the codling moth (Cydia pomonella), striped cucumber beetle (Acalymma vittatum), and the Japanese beetle (Popilla japonica). The two compounds were also significantly active i n the 9 K B (human nasopharyngeal carcinoma) cytotoxicity assay ( n e r i i f o l i n E D C Q 2 . 2 χ 1 0 mcg/ml and 2 ' - a c e t y l n e r i i f o l i n E D C Q 3 . 3 χ 1 0 mcg/ml) ( H ) . In mammals, these glycosides are cardiotoxic and are quite potent ( L D 5 0 i n cats, 0 . 1 9 6 mg/kg and 0 . 1 4 7 mg/kg, respectively); extrapolation to a 7 0 kg human would put the f a t a l dose at about 1 0 - 1 4 mg; as a comparison the f a t a l dose of hydrogen cyanide for humans i s 5 0 - 1 0 0 mg. However, i n human poisoning with cardiac glycosides, as found i i n red s q u i l l , emesis should prevent fatalities after oral ingestion. In our study, cardiac glycosides were proven to be very potent as natural insecticides, and t h i s protective e f f e c t l i k e l y explains the widespread d i s t r i b u t i o n of these glycosides i n several plant families.


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A U.S. patent application vas subsequently f i l e d to claim the use of these agents i n the control of insect pests (12), but the r e s u l t i n g publication ( H ) preceded the f i l i n g date of the patent application by a fev days over one year, and the patent vas disallowed. Nonetheless, ve remained convinced that these procedures could vork to y i e l d nev natural pesticides. Hovever, a reviev of the project suggested a major need for improvement. The corn borer bioassay vas too labor-intensive and i t rapidly depleted our extracts.


ERIPE SHRItt? PIQASSAY To facilitate continued research for such useful bioactive materials from higher plants, a convenient general bioassay vas needed. Specific bioassays, e.g., receptor binding, enzyme i n h i b i t i o n , etc., often overlook other useful a c t i v i t i e s vhich are not detected, or are ignored, i n the screening process. There i s a r e a l need for r e l i a b l e , general bioassays which can detect the broad spectrum of b i o a c t i v i t i e s present i n higher plant extracts and, yet, can be employed by natural product chemists, i n house, at lov cost, using a minimum amount of test materials, to permit phytochemical screening and to guide fractionation. Since most active plant principles are toxic at elevated doses, our approach has been to develop a general bioassay that simply screens for l e t h a l i t y i n the simplest of macroscopic zoologie systems. Once the active plant species have been detected, a battery of more expensive and specialized bioassays could then suggest the best candidates for large scale fractionation. Our premise here has been that toxicology i s simply pharmacology at a higher dose, or pharmacology i s simply toxicology at a lover dose. Thus, a general bioassay for l e t h a l i t y might lead to new useful pharmacologic agents. However, i f the agents prove to be persistently toxic, with narrow therapeutic indexes, and useless as drugs, we might then promote them as pesticides. After considering several possible organisms, we chose brine shrimp (Artemia salina), a tiny crustacean, as our general bioassay tool (13). The eggs of brine shrimp are r e a d i l y available at low cost i n pet shops as a food for t r o p i c a l f i s h . The eggs remain viable for years, especially i f refrigerated, i n the dry state. Upon being placed i n a "brine solution, the eggs hatch within 48 hours and swim toward a l i g h t source, providing large numbers of larvae ( n a u p l i i ) . Compounds and extracts are tested i n i t i a l l y at concentrations of 1 0 , 1 0 0 , and 1 0 0 0 ppm a f t e r being placed i n v i a l s containing 5 ml of bTine and ten shrimp i n each of three replicates. Survivors are counted a f t e r 24 hours, and the percentage of deaths a t each dose i s recorded. Intermediate dosages can be tested once the effective dosage range i s determined. These data can then be used, i n a simple program on an IBM personal computer, to estimate L C 5 0 values and 95% confidence iintervals. The L C ^ Q values, thus, give us a single value for the comparison of potencies among various extracts. These procedures are summarized i n Table I (14).


3. ALKOFAHI ET AL. Table I.


Materials and Procedures for Brine Shrimp Lethality Bioassay

A.

Materials 1.ArtemLa salina Leach (brine shrimp eggs from store) 2.Sea s a l t (from f i s h store) 3.Small tank with perforated dividing dam to grow shrimp, cover, and lamp to attract shrimp. 4.Syringes; 5 ml, 0.5 ml, 100 mcl, and 10 mcl. 5.2 dram v i a l s (9 per sample + 1 control)


B.

Procedures 1. Make sea water according to directions on box (ca. 38 g sea s a l t per l i t e r of water), f i l t e r . 2. Put sea water i n small tank and add shrimp eggs to one side of the divided tank, cover this side. Lamp on other side w i l l attract shrimp through perforations i n dam. 3. Allow 2 days for the shrimp to hatch and mature. 4. Prepare v i a l s f o r testing; f o r each fraction, test i n i t i a l l y at 1000, 100, and 10 mcg/ml; prepare 3 v i a l s at each concentration f o r a t o t a l of 9 v i a l s ; weigh 20 mg of sample and add 2 ml of solvent (20 mg/2 ml); from this solution transfer 500, 50, or 5 mcl to v i a l s corresponding to 1000, 100, or 10 mcg/ml, respectively. Evaporate solvent under nitrogen and then put under high vacuum for about 30 min; v o l a t i l e solvents w i l l evaporate over night. Alternatively, materials may be dissolved i n DMSO, and up to 50 mcl may be added/5 ml brine before DMSO t o x i c i t y affects the results. 5. After 2 days (when the shrimp larvae are ready), add 5 ml sea water to each v i a l and count 10 shrimp per v i a l (30 shrimp per dilution). 6.24 hours l a t e r count and record the number of survivors. 7. Analyze data with Finney computer program to determine L C ^ Q values and 95% confidence intervals. A copy of this program for IBM PC's i s available from Dr. McLaughlin. 8. Additional dilutions at less than 10 mcl/ml may be needed for potent materials; intermediate concentrations can be prepared and tested to narrow the confidence intervals.

Since 1982, the brine shrimp bioassay, combined with s p e c i f i c antitumor bioassays, has led us to several i n vivo active plant antitumor agents (15. 16 inter a l i o s ) . In addition, we have used this system i n the screening and fractionation of p e s t i c i d a l plant extracts as described below. I t has become the "work horse" bioassay of our laboratory. Each Investigator performs his/her own brine shrimp tests and quickly (24 hours) has the results. The results are quantitative and quite reproducible within the 95% confidence l i m i t s . Our contention i s that t o x i c i t y to this simple crustacean i s a convenient indicator o f t o x i c i t y to invertebrate economic pests.



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Plants and Extracts Involved i n Screening Our Department of Medicinal Chemistry and Pharmacognosy at Purdue has an established and continuing interest i n the chemistry of bioactive natural products. In recent years, we have focused primarily on antitumor compounds isolated and characterized from higher plants through the support of grants and contracts from the National Cancer Institute (NCI). In our warehouse now are varying quantities (0.5 - several kg) of over 2000 species of interesting higher plants. These, hereafter referred to as "warehouse plants" (Table I I ) , for the most part, do not represent random collections but have f o l k l o r i c or poisonous h i s t o r i e s , were l e t h a l to mice i n the NCI antitumor screen, were cytotoxic, or have other reasons for us to suspect that they may possess interesting b i o l o g i c a l activities. This c o l l e c t i o n represents thousands of dollars already spent i n acquisition, shipping, botanical authentication, drying, m i l l i n g , and storage. In addition, extracts of nearly 400 plant species were on hand from the National Cancer Institute (McCloud Collection) and through a collaborative project with King Saud University i n Riyadh, Saudi Arabia (Table I I ) . A continuing flow of new plant materials i s maintained through several international collaborators (Iran, China, India, S r i Lanka, Thailand, Panama, Jordan, Venezuela, B r a z i l ) . Screening of a l l these plant extracts was i n i t i a t e d i n a previous contract (1985-86) between E l i L i l l y (Greenfield) and Purdue University. Approximately f i v e new warehouse plant species per week were extracted, partitioned and screened. Screening data was obtained on extracts of 739 species. The extracts represent the three d i f f e r e n t sets of plant materials as summarized i n Table II. Extracts of the warehouse plants and a few of the most active extracts of the Saudi plants were partitioned through Scheme 1 to provide extracts F001-F006. Samples of approximately 100 mg of each i n i t i a l extract or partitioned f r a c t i o n were submitted, through weekly mailings at ca. 30/week, to Greenfield. At L i l l y , tests are conducted i n seven indicator organisms (mosquito larvae, blowfly larvae, Caenorhabditis elegans or Halmonchus contortus, corn rootworm, two-spotted spider mite, southern army worm, and melon aphid). The extracts of the active McCloud plants were also assayed for brine shrimp l e t h a l i t y i n our laboratory. Corn rootworm a c t i v i t y , at 300 ppm, was difficult to detect, and southern army worm (SAW), at even 5000 ppm, was resistant to a l l but a few species. The f r e e - l i v i n g nematode, C. elegans, gave too many positives i n the f i r s t year, and i n the second year was replaced by H. contortus which i s more s e l e c t i v e . Results of extraction and p a r t i t i o n i n g (Scheme 1) of Chrysanthemum flowers (containing pyrethrins) and Lonchocarpus roots (containing rotenone), as controls, demonstrated the effectiveness of these procedures at detecting such desirable a c t i v i t i e s and i n enriching the p e s t i c i d a l materials. As a further control, extracts of Warburgîa salutaris (W. ugandensis) (Canellaceae), screened as positive i n the p e s t i c i d a l assays and, by a c t i v i t y - d i r e c t e d fractionation monitoring with brine shrimp, yielded the expected drimane sesquiterpenes, warburganal and


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ALKOFAHI ET AL.

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Plant : genus

species

family

I d e n t i f i c a t i o n No.:

dried powdered plant material


(100 g) 95% EtOH

95% EtOH solubles ( g)

marc (Discard)

F001 saved g f o r testing; p a r t i t i o n between CHCI3/H2O (1:1)

water solubles ( g)

any insoluble interface ( g)

F002

F004

CHCI3 solubles ( g) F003 saved g for testing; p a r t i t i o n between hexane/90% aq. MeOH (1:1)

90% MeOH solubles ( g)

hexane solubles ( g)

F005

Submitted f r a c t i o n s ( testing; Date

F006

) to

for

Scheme 1 . Standard Flow Sheet of Extraction and I n i t i a l P a r t i t i o n i n g to Provide Screening Extracts f o r Bioassays



32 Table I I .

Sources of Plant Materials Screened

A. Warehouse Collection: (EtOH extracts a l l partitioned to F001-F006, see Scheme 1) 359 species (98 families) B. Saudi Collection: (EtOH or CHClo extracts; a few partitioned to give F001-F006), 136 species (43 families)

give

actives

C. McCloud Collection: (CH Cl2 extracts; none partitioned), (from NCI) 244 t o t a l species: 92 actives (46 families) and 152 inactives (56 families)


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muzigadial (1Z, 18) which are known to exhibit potent antifeedant (19-21) and molluscicidal (21) a c t i v i t i e s .

insect

The p e s t i c i d a l screening results were tabulated on computers both at Greenfield and i n our laboratory at Purdue. The computer programs permit tabulation of extracts and species active against s p e c i f i c pests or against various combination of pests. Choosing those with the broadest spectrum of a c t i v i t i e s , we selected twenty target plant species for large-scale, b i o a c t i v i t y - d i r e c t e d , fractionation. A l i t e r a t u r e search shows that, aside from our work, no previous b i o a c t i v i t y - d i r e c t e d research has been performed on these target species. One of the target species, the common paw paw, Asimina triloba, Annonaceae, has been a subject of our fractionation e f f o r t s for the past five years. This work has now been successful; the bioassay-directed fractionation has not been published previously, and w i l l be discussed below. Proprietary interests prevent us from disclosing additional target species and the screening r e s u l t s . THE PAW

PAW

PROJECT

Isolation Procedure Extraction of Plant Material. Bark of Asimina triloba (paw paw), collected at the Purdue Horticulture Farm, was dried and ground i n a Wiley m i l l to 2 mm. The ground bark (3.990 kg) was extracted by exhaustive percolation with 185 1 of 95% ethanol. The ethanol solubles were vacuum evaporated to a syrupy residue which was labeled Fraction F017. Fraction F017 was partitioned between QUC^/I^O (1:1), and the water solubles were taken to dryness and labeled F018. The C ^ C ^ solubles were recovered as a syrupy residue using a solvent evaporator. This residue was labeled Fraction F019, and a 223 g sample of F019 was then partitioned between hexane and 90% aqueous methanol. The methanol solubles were thereafter vacuum evaporated to a thickened syrup (41 g) and labeled as Fraction F020. The recovered hexane solubles constituted Fraction F021. The results of the brine shrimp bioassay on the fractions are reported i n Table I I I .


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A L K O F A H I ET AL.

Table III.

Assay of P a r t i t i o n Fractions from Ethanolic Extract of the Bark of Asimina triloba

Brine shrimp L C ^ Q (ppm)

Fraction No.

017 (ethanol extract) 018 (H 0 solubles) 019 (CH C1 solubles) 020 (aqueous methanol solubles) 021 (hexane solubles) asimicin 2


2

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Pesticides from Higher Plants

2

7.56 >1000 1.67 0.04 715 0.03

Chromatography of Fraction F020. A sample of F020 (39.5 g), the most toxic p a r t i t i o n fraction indicated by the brine shrimp assay (Table III), was adsorbed on c e l i t e (150 g) and applied to a s i l i c a column (4.0 kg) packed i n benzene/EtOAc (80:20). The column was eluted with 1 0 - l i t e r aliquots of 20%, 50% EtOAc/benzene, 100% EtOAc, 2%, 5%, 20%, 50% MeOH/EtOAc, and f i n a l l y , 100% MeOH, and fractions were dried and weighed. A TLC plate was run, i n hexane/EtOAc (20:80), on every f i f t h column f r a c t i o n and sprayed with 0.5% tetrazolium blue i n MeOH:5N NaOH (1:1). From the appearance after TLC, pools of similar compounds were made and the pools assayed with brine shrimp. The results are reported i n Table IV. C-18 Column Chromatography of Fraction AT 49. The most active f r a c t i o n i n the brine shrimp assay, AT 42, (Table IV) was consumed i n development of a satisfactory separation method. The next most active and comparable fraction, AT 49, was subjected to C-18 column chromatography; AT 49 (1.924 g) was adsorbed on c e l i t e (8 g), applied to the top of a column of C-18 s i l i c a (60 g), and chromatographed and assayed for brine shrimp l e t h a l i t y as shown i n Table V. Chromatotron Separation of Fraction AT 49-5. Separation of the most abundant active fraction AT 49-5, which was active i n the brine shrimp assay, from the above C-18 column separation of AT 49 (Table V) was carried out as follows. A 4 mm s i l i c a chromatotron rotor was loaded with 586.3 mg of AT 49-5 and eluted with CHCl /MeOH/H 0 (5:2:2); this resulted i n 25 fractions which were then pooled on the basis of their s i m i l a r i t i e s upon TLC analysis (Table VI). 3

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P u r i f i c a t i o n of Fraction AT 49-5-2. A white waxy substance (m.p. 68x-69x C) precipitated from an E t 0 solution at AT 49-5-2 upon the addition of hexane. The precipitate was collected, assayed i n five different TLC systems, and v i s u a l i z e d with N a C r 0 i n 40% H S0^. Each of the TLC analyses showed only a single spot 2

2

2


2

7


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Table IV. Toxicity of Column Chromatography Fractions from F020 (39.5 g) (see Table III) to Brine Shrimp


Pool No.

Fractions pooled

1-5 6-10 11-15 16-22 23-25 26-32 33-36 37-42 43-49 50-55 56-58 59-67 68-71 72-74 75-80 81-88 89-93 94-98 99-121

AT 5 AT 10 AT 15 AT 22 AT 25 AT 32 AT 36 AT 42 AT 49 AT 55 AT 58 AT 67 AT 71 AT 74 AT 80 AT 88 AT 93 AT 98 AT 121

Weight (g)

2.71 1.52 0.64 0.35 0.33 2.70 2.04 2.31 2.33 5.12 0.66 1.59 0.45 2.46 2.86 0.47 0.38 2.59 6.12

Brine shrimp LC (mcg/ml) 5 0

>200 >200 >200 >200 >200 >200 0.15 0.07 0.13 0.85 1.07 0.33 0.49 0.52 0.37 0.52 0.46 >200 >200

Table V. Toxicity of Column Chromatography Fractions from AT 49 (1.924 g) (see Table IV) to Brine Shrimp

Solvent

Weight (mg)

50% MeOH (100 ml) 60% MeOH (100 ml) 70% MeOH (100 ml) 80% MeOH (100 ml) 90% MeOH (100 ml) MeOH (100 ml) MeOH (100 ml)

36. 6 30. 8 28..9 49,.2 832 .3 433 .5 363 .8

Fraction No.

Brine Shrimp LC (mcg/ml)

AT 49-1 AT 49-2 AT 49-3 AT 49-4 AT 49-5 AT 49-6 AT 49-7

0.30 6.28 4.,92 0,37 0..36 0..36 1 .00

5 0


3. ALKOFAHI ET AL. Table VI.

Chromatotron Separation of AT 49-5

Pool No.


AT AT AT AT AT


49-5-1 49-5-2 49-5-3 49-5-4 49-5-5

Fractions

35 (see Table V)

Weight (mg)

25..9 342. .1 136, .7 58 .3 20 .4

1-6 7-13 14-16 17-20 21-25

indicating homogeneity. The collected compound, labeled AT-II (81.35 mg), was then subjected to structural analyses, including: high resolution FAB ms, i r , uv, H nmr (CDCI3) with selective τϊ- H decouplings, and C nmr . This compound was named "asimicin" and was assigned the structure indicated. The structural elucidation of asimicin, without stereochemistry, has been published (21); however, i t has eight c h i r a l centers. We have recently defined the stereochemistry at Cjc ^ ^ 20 23 24 / t / t h / t / t h by comparing H nmr values of the 'triace'tat'e with a series of reference acetylated bis-tetrahydrofurans (24). The configuration at C^ and Cog i s suggested to be S and R, respectively, by cd and comparison with r o l l i n i a s t a t i n (Hui, Y.-H.; Rupprecht, J.K.; Anderson, J.E.; Liu, Y.-M.; Smith, D.L.; C.-j. Chang; McLaughlin, J.L. J . Nat. Prod.. submitted for publication). a

s

t n

BIOASSAYS OF PAW PAW FRACTIONS AND ASIMICIN Fractions F017, F020, AT 36, and AT 49 from the fractionation of AsLminia triloba bark extract were bioassayed at various levels of test material with several common agronomic pests. The southern armyworm {Spodoptera eridania) bioassay was conducted by f i r s t applying an aqueous solution of 5000 ppm test material to leaves of a squash plant and allowing the leaves to dry. The leaves were then removed from the plant and placed i n p e t r i dishes with the armyworm larvae. The percent mortality was computed from the number of dead larvae after 3 days. The effectiveness of the test fractions against the two spotted spider mite (Tetranychus urticae), an arthropod, and the melon aphid (Aphis gossypii) was determined by spraying infested leaves of squash plants with aqueous solutions containing 4000 ppm test material and observing the percent mortality after 24 hrs. The assay with yellow fever mosquito larvae, Aedes aegypti, involved suspending the larvae i n an aqueous solution of the test



36


material, at concentrations of 1000 ppm and less, and determining the percent mortality after 24 hrs. For assaying with the southern com rootworm, Diabrotica undecimpunctata, s o i l samples treated with 300 ppm of test material were placed i n conical paper cups having a small opening at the lower apical end. Larvae emerging from the opening were evaluated for percent mortality after 3 days. In the corn leafhopper (Dalbulus maidis) assay, f i l t e r paper i n the bottom of a p e t r i dish was wetted with the test solution and allowed to dry. Thereafter, the leafhoppers and a measured amount of food were introduced to the dish, and the percent mortality was determined after 24 hrs. The comparative results of the screening of Fractions F017, F020, AT 36, and AT 49 (see Tables III and IV) against the aforementioned test organisms are reported in Table VII. Significant a c t i v i t i e s were detected against melon aphid and mosquito larvae, some a c t i v i t y was apparent against two spotted spider mites and corn leafhoppers, and no a c t i v i t y was observed against southern armyworm and corn rootworm. Fractions F017, F018, F019, and F020, from the fractionation of Asimina triloba bark extract (see Table III) were bioassayed with mosquito larvae, southern corn rootworm, southern armyworm, two spotted spider mite, and melon aphid. Generally, brine shrimp l e t h a l i t y paralleled the i n s e c t i c i d a l a c t i v i t i e s . In addition, these fractions were assayed with blowfly larvae (Colliphora vicina) and the nematode (Caenorhabditis elegans). The blowfly larvae assay was conducted by dipping a gauze dental wick into bovine serum containing 1% (w/v) test material. The larvae were thereafter introduced to the wick and evaluated for percent mortality after 24 hrs. The nematode (C. elegans) bioassay was conducted by suspending the worms i n a 0.1% (w/v) aqueous solution of the test material and determining the percent mortality after 3 days. The results of this bioassay series are reported i n Table VIII. The a c t i v i t i e s of Fractions F020, AT 49, and p u r i f i e d asimicin were compared to the commercial pesticides pyrethrum (57%) and rotenone (97%) i n assays with the Mexican bean beetle (Epilachna varivestis), the melon aphid, mosquito larvae, the nematode C. elegans, and blowfly larvae. The Mexican bean beetle assay was conducted by spraying the material onto the bean leaves, allowing the leaves to dry, and then introducing the third-instar beetles to the leaves i n an enclosed chamber. After 72 hrs., the percent mortality was determined. The assays on the remaining systems were conducted as previously described. The results are reported i n Table IX. Asimicin isolated from the seeds of Asimina triloba was assayed by two separate methods against the striped cucumber beetle (Acalymma vittatum) as described below. Two-Choice Leaf Disc Bioassavs. Appropriate quantities of the test material were suspended i n acetone, diluted with water containing 0.01% of Tween 20 to a sample concentration of 0.5% (w/v), and then the suspension was emulsified with a Brinkman Polytron homogenizer. The 0.1% solutions were derived by d i l u t i o n of the 0.5% mixture. Leaf discs (2.0 cm diameter) cut from BURPEE HYBRID



3.

ALKOFAHI

ET A L .


37

cantaloupe leaves were dipped i n either the sample homogenate or a corresponding homogenate containing no sample (control discs), a i r dried, and then two of each type of disc were arranged alternately around 93 mm diameter χ 73 mm deep polyethylene dishes. Five newly emerged female striped cucumber beetles, after being starved for 24 hrs, were introduced into each chamber, and the chambers were covered with muslin; these covers were kept moist during the f i r s t 6 hrs of the bioassay by misting them p e r i o d i c a l l y with water. Tests were conducted i n two replicates under ambient greenhouse conditions. Observations were made at 3, 6, and 22 hrs to estimate v i s u a l l y the amount of leaf tissue consumed and to check f o r deaths. Data are presented (Table X) as a consumption index, which i s defined as percentage of treated discs consumed χ 100/(percentage of control discs consumed+percentage of treated discs consumed). A value of 50 indicates treated and untreated discs have been consumed i n equal amounts; an extract that gives an index of 20 or less i s considered highly deterrent i n these bioassays. No-Choice Leaf Disc Bioassav. For the no-choice feeding study, homogenate samples preparation (0.5% w/v solutions only), leaf disc (1.5 cm diameter) treatment and drying, and insect preparation were as described f o r the two-choice bioassay. Single treated discs were placed i n individual 2-dram glass v i a l s , and one beetle was introduced into each v i a l . Water was provided by a soaked 0.5 cm length of dental wick, and the v i a l s were stoppered with cotton plugs. The bioassays were conducted i n 10 replicates under ambient greenhouse conditions; leaf consumption and mortality data were taken daily f o r 3 days. Data are presented as percent of leaf discs consumed. The results of the two-choice and no-choice leaf disc bioassays are reported i n Tables X and XI. COMMERCIAL POTENTIAL The chemical synthesis of asimicin would be d i f f i c u l t (there are 256 isomers); thus, a crude extract of the plant material, l i k e l y the bark of Annonaceous trees, might furnish an economical source of such acetogenins as pesticides. F020 from the paw paw bark i s the most potent of the crude extracts (Tables I I I , VII, VIII, and IX) ; a f r a c t i o n similar to F020 could be readily prepared from the dried, bioactive, bark of a number of Annonaceous trees. The extracts could be standardized by a bioassay, such as brine shrimp l e t h a l i t y , to provide a consistent product. Chemically F020 from paw paw contains asimicin plus at least five additional acetogenins three o f which have been isolated and remain to be chemically characterized. We have bioassayed bark extracts of numerous Annonaceae species and f i n d similar a c t i v i t y i n many members of this family. TLC suggests that acetogenins are commonly present i n the active species. In a preliminary f i e l d test, F020 was suspended i n 0.5 or 1% (5,000 or 10,000 ppm) aqueous solutions using 2 or 3% Tween 80 as the suspending agent. The solutions were sprayed onto rows of green bush beans (blue lake variety) which were experiencing a natural infestation with bean leaf beetles (Cerotoma trifurcata).



38 Table VII.

Assay of Paw paw (Asimina triloba) Toxicity to Agronomic Pests

Fractions for

% Mortality

Fraction

Southern armyworm

200 20 12 200 20 12 200 100 20 12 200 100 20 12

F017


Concentration (ppm)

F020

AT 36

AT 49

Two spotted spider mite

Melon aphid

Mosquito larvae

0

10

50 30 100 10

90 100

10

90 100

% Mortality

Fraction

F017

F020

AT 36

AT 49

a

"

Concentration (ppm)

200 20 12 200 20 12 200 100 20 12 200 100 20 12

Southern corn rootworm

Corn leafhopper

_ a

—0 — —0 — — —0 — — —0

— — — — — — 10 — — —0 ---

" indicates not tested.


ALKOFAHI ET AL.

39


Table VIII. Toxicity Assay of Paw paw (Asimina triloba) Fractions (Table III) with Representative Agronomic Pests


% Mortality

Fraction

Mosquito larvae 1000 100 10 1 ppm ppm ppm ppm

F017 F018 F019 F020

100 50 100 100

90 0 100 100

0 0 70 100

0 0 0 80

Blowfly larvae 1%

C. elegans 1%

0 0 100 100

100 0 100 100

Southern corn rootworm 300 ppm

% Mortality Southern Fraction

F017 F018 F019 F020

Two spotted Melon armyworm spider mite aphid 5000 ppm 5000 ppm 5000 ppm

0 0 0 0

30 0 40 60

0 0 20 50


40

INSECTICIDES O F PLANT ORIGIN

Table IX. Comparative A c t i v i t i e s of Paw paw (Asimina triloba) Fractions, Asimicin, and Standard Insecticides

% Mortality


Treatment material

Rate ppm

Mexican bean beetle 72 hr

F020 5000 (Table III) 1000 500 100 10 1 5000 AT 49 (Table IV) 1000 500 100 10 1 Asimicin 1000 (purified) 500 100 50 10 1 0. 1 Pyrethrum 500 (57% pure) 100 50 10 Rotenone 1000 (97% pure) 500 100 10 1

a

"—"

Melon aphid 24 hr

100 100 100 60

80 0 0 0

— — 100

— — 90

100 100 100

0 0 0

— — — 100 100 100 70 0 100 100 100 0

— — — — —

100 20 0 0 0 100 100 100 20 0

— — —

0

Mosquito larvae 24 hr

Nematode 72 hr

Blowfl] larvae 24 hr

...

...

...

1Ό©

100

100

—

—0

—

10® 80 10

100 100 0

100

100

—

100

— 100

— 100

—0

100 50

100 100

— —

100

—0

—

—0

Ό

100 100 100

100

—

—0

— — 100

100

—

—0

— — 100

— —0

100

—

100

50 0

— —

— —

indicates not tested.


0

—

-

— 100

0

—

0 100

— — — —

3.

ALKOFAHI ET AL.

41


After three applications, within a period of 10 days, there was no v i s i b l e damage to the new leaves of the test row and i t was several inches higher than the control row. The a c t i v i t y seems to remain stable i n sunlight. Mechanism of action and toxicology studies remain to be completed. A U.S. patent has been granted to us covering the use of these Annonaceous acetogenins i n pest control (2£) · A d i v i s i o n a l patent on the composition of matter of asimicin i s pending. We hope to Table X.

Striped Cucumber Beetle Two-Choice Assay for Asimicin


Consumption index Asimicin concentration, %

0.1 0.5

3 hr

6 hr

25 0

20 0

22 hr

14 0

a

Value of 50 means that equal amounts of treated and control discs were consumed. Value of 0 means that none of the treated disc was consumed.

Table XI.

Striped Cucumber Beetle No-Choice Assay for Asimicin

Mortality, %

Leaf consumed, %

Treatment

1 day

2 days

Asimicin (0.5%) Control

0

0

31

59

1 day

2 days

3 days

0

40

50

50

74

0

0

0

3 days

license this discovery as an " a l l natural" garden pesticide. While our patent was being processed, another U.S. patent (26) was issued to the Bayer Co. i n Germany; their patent protects the i n s e c t i c i d a l use of an unknown substance, c a l l e d "annonin", isolated from Annona squamosa (Annonaceae). CONCLUSIONS The screening of higher plants for novel pesticides detects interesting active leads. The a c t i v i t y - d i r e c t e d fractionation of seeds of Thevetia thevethioides (Apocynaceae), using European corn


INSECTICIDES O F PLANT ORIGIN

42


borers, l e d us to two known cardiac glycosides, n e r i i f o l i n and 2'acetylneriifolin. The a c t i v i t y - d i r e c t e d fractionation of seeds and bark of Asimina triloba (Annonaceae) (paw paw) l e d us to a mixture of potent novel acetogenins of which asimicin has been characterized. These acetogenins are common to a large number of Annonaceae. Commercial development of the Annonaceous extracts as a garden pesticide seems feasible provided that the materials prove safe. In addition to being p e s t i c i d a l , we have determined that asimicin has additional b i o l o g i c a l effects which may be worthy of further study. The fractionation of target plant species i n other families should provide more of such new compounds f o r p r a c t i c a l exploitation.

LITERATURE CITED 1. Jacobson, M. Insecticides of the Future; Marcel Dekker: New York, 1975. 2. Mandava, N.B., ed. Handbook of Natural Pesticides; Methods; CRC Press: Boca Raton, FL, 1985; Vols. 1 and 2. 3. Leahey, J.P. The Pyrethroid Insecticides; Taylor and Francis, Inc.: Philadelphia, 1985. 4. Jacobson, M.; Crosby, D.G. Naturally Occurring Insecticides; Marcel Dekker: New York, 1971. 5. Balandrin, M.F.; Klocke, J.Α.; Wurtele, E.S.; Bollinger, W.H. Science 1985, 228, 1154. 6. Schroeder, D.R.; Nakanishi, K. J . Nat. Prod. 1987, 50, 241. 7. Jacobson, M. Insecticides from Plants, A Review of the Literature, 1941-1953; USDA Agric. Handb. 154, 1958; p 299. 8. Jacobson, M. Insecticides from Plants, A Review of the Literature, 1954-1971; USDA Agric. Handb. 461, 1975; p 138. 9. Guthrie, W.D.; Raun, E.S.; Dicke, F.F.; Pesko, G.R.; Carter, S.W. Iowa State J. Sci. 1965, 40, 65. 10. Freedman, B.; Nowack, L . J . ; Kwolek, W.F.; Berry, E.C.; Guthrie, W.D. J. Econ. Entomol. 1979, 72, 541. 11. McLaughlin, J . L . ; Freedman, B.; Powell, R.G.; Smith, C.R., Jr. J . Econ. Entomol. 1980, 73, 398. 12. Freedman, B.; McLaughlin, J . L . ; Powell, R.G., Smith, C.R., Jr.; Reed, D.K.; Ledd, T.L., Jr. Control of Insect Pests with Neriifolin and 2'-Acetylnerrifolin; Serial No. 061289,948 filed for U.S. Patent Aug. 4, 1981; abandoned June 29, 1982. 13. Jaspers, Ε., ed. Fundamental and Applied Research on The Brine Shrimp Artemia salina (L.) in Belgium; European Mariculture Society, Special Publication No. 2: Bredene, Belgium, 1977. 14. Meyer, B.N.; Ferrigni, N.R.; Putnam, J . E . ; Jacobsen, L.B.; Nichols, D.E.; McLaughlin, J.L. Planta Medica 1982, 45, 31. 15. Anderson, J . E . ; Chang, C . - j . ; McLaughlin, J.L. J . Nat. Prod. 1988, 51, 307. 16. Alkofahi, Α.; Rupprecht, J.K.; Smith, D.L.; Chang, C.-j.; McLaughlin, J.L. Experientia 1988, 44, 83. 17. Kubo, I.; Lee, Y .-W.; Pettei, M.; Dilkiewicz, F.; Nakanishi, K. J.C.S. Chem. Comm. 1976, 1013. 18. Nakanishi, K.; Kubo, I. Israel J. Chem. 1977, 16, 28.


3. ALKOFAHI ET AL.


19. Kubo, I.; Miura, I.; Pettei, M.; Lee, Y.-W.; Dilkiewicz, Nakanishi, K. Tetrahedron Lett. 1977, 52, 4553. 20. Kubo, I.; Matsumoto, T.; Kakooko, Α.; Mubiru, N. Chem. Lett. 1983, 979. 21. Nakata, T.; Akita, H.; Naito, T.; Oishi, T. J. Am. Chem. Soc. 1979, 101, 4400. 22. Okawara, H.; Nakai, H.; Ohno, M. Tetrahedron Lett. 1982, 1087. 23. Rupprecht, J.K.; Chang, C.-j.; Cassady, J.M.; McLaughlin, J.L.; Mikolajczak, K.L.; Weisleder, D. Heterocycles 1986, 24, 1197. 24. Hoye, T.R.; Sukadolnik, J.C. J . Am. Chem. Soc. 1987, 109, 4402. 25. Mikolajczak, K.L.; McLaughlin, J . L . ; Rupprecht, J.K. U.S. Patent 4 721 729, issued Jan. 26, 1988. 26. Moeschler, H.G.; Pfluger, W.; Wendisch, D. U.S. Patent 4 689 232, issued Aug. 25, 1987. F.;


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November 28, 1988


Insecticides of Plant Origin - ACS Publications - American Chemical

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